CN115152668B - Production method of tetraploid culter ilishaeformis - Google Patents

Production method of tetraploid culter ilishaeformis Download PDF

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CN115152668B
CN115152668B CN202210725611.7A CN202210725611A CN115152668B CN 115152668 B CN115152668 B CN 115152668B CN 202210725611 A CN202210725611 A CN 202210725611A CN 115152668 B CN115152668 B CN 115152668B
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ilishaeformis
tetraploid
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CN115152668A (en
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薛淑群
陈伟兴
夏邦华
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Northeast Agricultural University
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K61/00—Culture of aquatic animals
    • A01K61/10—Culture of aquatic animals of fish
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K61/00—Culture of aquatic animals
    • A01K61/10—Culture of aquatic animals of fish
    • A01K61/17—Hatching, e.g. incubators
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
    • Y02A40/81—Aquaculture, e.g. of fish

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  • Marine Sciences & Fisheries (AREA)
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Abstract

A production method of tetraploid culter ilishaeformis belongs to the technical field of fish breeding of subfamily culter. In order to solve the technical problem of directly obtaining triploid fishes, the invention establishes a production method suitable for tetraploid ilishaeformis, and the method adopts a mode of combining hydrostatic pressure treatment, temperature shock, chemical substance combined induction treatment and chemical substance induction treatment to determine the technical parameters of the first cleavage of fertilized eggs of the ilishaeformis and the optimal production conditions of tetraploid induction for the first time. The tetraploid culter ilishaeformis production method has clear operation flow and strong practicability, can successfully induce the tetraploid culter ilishaeformis to be produced, has the induction efficiency reaching 23 percent, is suitable for the production of artificially induced tetraploid culter ilishaeformis, opens up a new technical path for polyploid breeding of the culter ilishaeformis, and provides data support for the rapid identification of the tetraploid culter ilishaeformis.

Description

Production method of tetraploid culter ilishaeformis
Technical Field
The invention belongs to the technical field of fish breeding in the subfamily culter, and particularly relates to a production method of tetraploid Erythroculter ilishaeformis.
Background
Culter ilishaeformis (culter alburnus), belonging to the order Cyprinus, family Cypriidae, subfamily culter, genus culter. The culter ilishaeformis is distributed in the dry, tributary and the subsidiary lakes of the water systems such as the Heilongjiang, the Yangtze river, the Zhujiang and the like, and is a precious economic fish in the places such as the middle and downstream of the Yangtze river, the Heilongjiang river basin and the like. The culter ilishaeformis has white and tender meat quality, delicious taste, higher nutritive value and insufficient market supply.
Gonad development affects the growth and meat quality of fish, reducing the commercial quality of fish. Along with the amplification of the culter ilishaeformis cultivation area, the probability of escaping into the nature is greatly increased, and pollution can be caused to fish germplasm resources in the nature. The triploid fish has unbalanced chromosome composition and gonad incapable of developing and maturing, so that the triploid fish has faster growth and higher commodity value than diploid fish, and is popular with consumers. From a cytogenetic perspective, triploids are an uneven chromosome set, thus preventing normal meiosis of germ cells, and often resulting in failure of gonads to develop. The sterile triploid economic fish has the following advantages because the energy loss of gonad development in the sexual maturity stage is avoided: 1. the quality of fish meat is prevented from being reduced in gonad development stage and spawning season, and the time to market is prolonged; 2. the growth arrest and the mortality increase in the gonadal development stage are avoided; 3. shortens the cultivation period, reduces the cultivation cost and can be cultivated into large individuals. Most fish in nature exist in diploid form except for few species, and offspring are propagated in an amphoteric fusion mode, and manual induction is needed to generate triploid. Polyploid events are rare in vertebrates compared to plants. The polyploidization event is an important impetus for species adaptive evolution. Polyploids containing multiple subgenomic sets often have the advantage of greater environmental flexibility. The fish chromosome has larger plasticity and is easy to double, which is the theoretical basis of the artificial induced polyploidy technical method. The triploid obtained by artificial means can be obtained in two ways. One is the direct route, i.e., treating fertilized eggs by physical or chemical means, impeding the second polar row. The physical method and the chemical method induce triploid, and can lead to the phenomena of high fertilized egg necrosis rate, low emergence rate, high juvenile fish deformity rate, low triploid rate and the like due to external stress and stress, so that the production scale of the triploid fish is greatly limited. The other is an indirect way, namely, tetraploid is obtained by first inducing, and then the tetraploid is naturally mated with diploid individuals after the tetraploid is sexually mature, so as to obtain triploid. In theory, the second approach has the advantage that once tetraploid individuals are obtained, physical or chemical induction is not needed, and the damage to embryos caused by the induction process is avoided. It becomes a very easy matter to obtain triploid if stable tetraploid lines can be established. However, most of the current manually induced triploid is obtained by the first approach, because the method of preventing the second pole efflux is relatively mature. The tetraploid induction is to prevent the first cleavage, which is much more difficult than the induction of triploid, and has low success rate, and the supermature tetraploid is obtained only in few fishes such as rainbow trout and crucian carp at present. The polyploid induction technology is researched, the tetraploid fish line with stable inheritance and extremely high purity is established, and the method has important practical significance and great application value for promoting the development of triploid culture industry.
The method for artificially inducing polyploidy mainly comprises the following steps: physical, chemical and biological methods. The physical methods mainly include hydrostatic pressure, temperature shock and electric shock, and the like, and the temperature shock and hydrostatic pressure are used in many cases. Whichever method is used for induction, the start time and duration of the treatment must be tightly controlled. Such as: taking a crystal color crucian carp as an example, the pressure sensitive period of fertilized eggs is only 4-5 minutes after fertilization, and the pressure is applied to destroy the activation and trimming processes of the eggs before the fertilized eggs start, so that development is blocked; followed by a stress refractory period, at which the second meiosis tends to be bright, the stress losing effect on the retained second diode. Therefore, in order to obtain triploid crystal color crucian carp, hydrostatic induction can only be performed 4-5 minutes after fertilization. Triploid induction results for salmon and clarias fuscus indicate that different initiation times and durations of induction treatments lead to different results.
So far, research on production methods of Guan subfamily tetraploid fishes and the like has not been reported. Due to the species difference, when the reported technical parameters of tetraploid induction of sea fish, crustaceans and most freshwater fish are used for tetraploid induction of the culter ilishaeformis, the induction rate of the tetraploid culter ilishaeformis is extremely low, and the production requirement is difficult to meet. Thus, there is a need to establish a production method suitable for tetraploid culter ilishaeformis.
Disclosure of Invention
In order to solve the technical problem of directly obtaining triploid fishes, the invention provides a production method suitable for tetraploid culter ilishaeformis, and the method can effectively induce the fertilized egg chromosome of the culter ilishaeformis to double to generate tetraploids, so that the fertilized egg chromosome is naturally mated with diploid individuals, and the triploid is easier to generate.
The production method of the tetraploid culter ilishaeformis provided by the invention specifically comprises the following steps:
(1) Artificial insemination of culter ilishaeformis: collecting mature eggs and semen of the culter ilishaeformis, placing the eggs in a drying container, slowly adding the semen, performing dry fertilization, slowly operating to uniformly mix the semen and the eggs, then adding culture water, and slowly shaking to complete the insemination process of the semen and obtain fertilized eggs;
(2) Inducing tetraploid culter ilishaeformis: placing the fertilized eggs obtained in the step (1) into a hydrostatic press bin, carrying out hydrostatic pressure treatment, carrying out temperature shock and cytochalasin B aqueous solution combined induction treatment on the fertilized eggs subjected to hydrostatic pressure treatment, taking out, transferring the fertilized eggs into the cytochalasin B aqueous solution for chemical induction, and finally transferring the fertilized eggs into culture water for hatching.
Further defined, the volume ratio of semen to eggs in step (1) is 1000. Mu.L:100 mL.
Further defined is that the temperature of the drying vessel of step (1) is from 23 to 25 ℃.
Further defined, the temperature of the water for cultivation in the step (1) is 23-25 ℃, and the addition amount is 10 times of the egg volume.
It is further defined that the hydrostatic pressure treatment of step (2) is 35 minutes after insemination.
Further defined, the hydrostatic pressure treatment of step (2) is performed at a pressure of 1200PSI for a treatment time of 3 minutes.
It is further defined that the combined induction treatment of the temperature shock and cytochalasin B aqueous solution in the step (2) is to immerse fertilized eggs into the cytochalasin B aqueous solution with the temperature of-2-2 ℃ and the concentration of 1.5 mg/L.
Further defined, the time of the combined induction treatment of the temperature shock and cytochalasin B aqueous solution in the step (2) is 10min.
Further defined, the chemical induction treatment of step (2) is incubating fertilized eggs in 1.5mg/L aqueous cytochalasin B solution at 23-25℃for 1h.
Further defined, incubating the culture water in step (2), wherein the culture water Shui Wenheng is set at 23-25 ℃.
The invention has the beneficial effects that:
the invention establishes a production method of the tetraploid culter by combining hydrostatic pressure treatment, temperature shock and chemical substance combined induction treatment with chemical substance induction treatment, and determines the technical parameters of the first cleavage of fertilized eggs of the culter and the optimal production conditions of tetraploid induction for the first time. The tetraploid culter ilishaeformis induction method established by the invention has clear operation flow and strong practicability, can successfully induce the tetraploid culter ilishaeformis, and has the induction efficiency of 23 percent. The production method of the tetraploid culter ilishaeformis is suitable for the production of the artificially induced tetraploid culter ilishaeformis, and opens up a new technical approach for polyploid breeding of the culter ilishaeformis.
The invention discovers that the tetraploid culter obtained by the tetraploid culter production method has obvious growth compared with the diploid culter, the area and the volume of the red blood cells are obviously increased, the multiple of the chromosome is increased, the content of the DNA in the cells is correspondingly increased, the volume of the cells is in direct proportion to the multiple of the chromosome, the structure of the testis tissue is more compact, and the data difference accords with the ploidy relation.
In addition, the identification process of the invention determines the length ratio and the weight ratio of the diploid Erythroculter ilishaeformis and the tetraploid Erythroculter ilishaeformis under the same culture condition; tetraploid culter erythrocyte volume and erythrocyte volume range; determining the ratio of the relative DNA content of the diploid culter ilishaeformis and the tetraploid culter ilishaeformis, and determining the absolute DNA content of the tetraploid culter ilishaeformis; determining the chromosome number of the tetraploid culter ilishaeformis; the difference between diploid and tetraploid Erythroculter ilishaeformis gonad sections is determined, and data support is provided for rapid identification of the tetraploid Erythroculter ilishaeformis.
Drawings
FIG. 1 is a graph showing the relative DNA content of diploid Erythroculter ilishaeformis;
FIG. 2 is a graph showing the relative DNA content of the tetraploid culter ilishaeformis;
FIG. 3 is a metaphase diagram of a diploid Erythroculter ilishaeformis chromosome;
FIG. 4 is a metaphase diagram of the tetraploid culter ilishaeformis chromosome;
FIG. 5 is a drawing of a 4-year-old diploid Erythroculter ilishaeformis testis tissue section (40X);
FIG. 6 is a drawing (40X) of a 4-year-old tetraploid culter ilishaeformis gonadal tissue section.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be further described in detail with reference to the following detailed description and the accompanying drawings. The experimental methods used in the examples below were conventional, and the materials, reagents, methods and apparatus used, unless otherwise indicated, were all conventional in the art and commercially available to those skilled in the art.
The invention compares the proportions of the tetraploid culter ilishaeformis obtained under different induction conditions, optimizes the production method suitable for the tetraploid culter ilishaeformis, and in all the comparative examples, the artificial insemination method of the culter ilishaeformis comprises the following steps:
the method comprises the steps of collecting mature eggs and semen after selective maturation of the culter ilishaeformis, placing the eggs in a drying container at 23-25 ℃, slowly adding the semen with the volume ratio of 1000 mu L to 100mL, performing dry fertilization, slowly operating to mix the semen and the eggs uniformly, then adding culture water at 23-25 ℃ which is 10 times of the egg volume, wherein the culture water meets the fishery water quality standard (GB 11607-1989), and slowly shaking to complete the insemination process of the semen and the eggs.
1. Determination of the method for inducing the tetraploid Erythroculter ilishaeformis
(1) Determination of the quadrupling start time:
comparative example 1: after fertilized sperm eggs for 30min, the hydrostatic pressure is 1000PSI, the acting time is 4min, and the sperm eggs are transferred into culture water to complete hatching.
Comparative example 2: after fertilization of sperm eggs, the sperm eggs are transferred into culture water to complete hatching under the hydrostatic pressure of 1000PSI for 4 min.
Comparative example 3: after fertilized sperm eggs, the sperm eggs are transferred into culture water to complete hatching under the hydrostatic pressure of 1000PSI for 4 min.
Comparative example 4: 45min after fertilization of sperm eggs, hydrostatic pressure of 1000PSI, action time of 4-5min, and transferring into culture water to complete hatching.
Comparative example 5: after fertilization of sperm eggs, the sperm eggs are transferred into culture water to complete hatching, wherein the hydrostatic pressure is 1000PSI, and the action time is 4-5 min.
The tetraploid proportion obtained in the above comparative example was counted, the tetraploid proportion of comparative example 1 was 2%, the tetraploid proportion of comparative example 2 was 8%, the tetraploid proportion of comparative example 3 was 5%, the tetraploid proportion of comparative example 4 was 0%, the tetraploid proportion of comparative example 5 was 0%, and the tetraploid proportion of comparative example 2 was the highest, and thus it was determined that the initiation time of inducing tetraploid was 35min after fertilization.
(2) Determination of hydrostatic pressure treatment time:
comparative example 6: 35min after fertilization, hydrostatic pressure of 1000PSI, action time of 2min, and transferring into culture water to complete hatching.
Comparative example 7: 35min after fertilization, hydrostatic pressure of 1000PSI, action time of 3min, and transferring into culture water to complete hatching.
Comparative example 2: 35min after fertilization, hydrostatic pressure of 1000PSI, action time of 4min, and transferring into culture water to complete hatching.
Comparative example 8: 35min after fertilization, hydrostatic pressure of 1000PSI, action time of 5min, and transferring into culture water to complete hatching.
The tetraploid proportion obtained in the above comparative example was counted, and it was found that the tetraploid proportion of comparative example 7 was 12%, and the tetraploid proportion was highest among several comparative examples, so that the time of hydrostatic pressure treatment was determined to be 3min.
(3) Determination of pressure during hydrostatic pressure treatment:
comparative example 9: 35min after fertilization, hydrostatic pressure 500PSI, action time 3min, and transferring into culture water to complete hatching.
Comparative example 10: 35min after fertilization, hydrostatic pressure 800PSI, action time of 3min, and transferring into culture water to complete hatching.
Comparative example 7: 35min after fertilization, hydrostatic pressure of 1000PSI, action time of 3min, and transferring into culture water to complete hatching.
Comparative example 11: 35min after fertilization, hydrostatic pressure of 1200PSI, action time of 3min, and transferring into culture water to complete hatching.
Comparative example 12: 35min after fertilization, the hydrostatic pressure is 1500PSI, the acting time is 3min, and the culture water is transferred to complete incubation.
Comparative example 13: 35min after fertilization, the hydrostatic pressure is 2000PSI, the acting time is 3min, and the culture water is transferred to complete incubation.
The tetraploid proportion obtained in the above comparative examples was counted, and the highest tetraploid proportion in each comparative example was comparative example 11, and the tetraploid proportion was 15%, so that the induction condition was determined to be 35min after fertilisation of sperm eggs, and the sperm eggs were treated for 3min under a hydrostatic pressure of 1200PSI, and transferred into aquaculture water to complete hatching.
(4) Determining whether to perform chemical induction and the chemical induction treatment time and the concentration of an inducer:
comparative example 11: 35min after fertilization, the hydrostatic pressure is 1200PSI, the acting time is 3min, after hydrostatic pressure treatment, fertilized eggs are not subjected to chemical substance induction treatment, and the fertilized eggs are transferred into culture water to complete hatching.
Comparative example 14: fertilize for 35min, hydrostatic pressure 1200PSI, action time is 3min, after hydrostatic pressure treatment, fertilized egg is treated by chemical induction, fertilized egg is immersed into 1.5mg/L cytochalasin B aqueous solution for treatment, cytochalasin B aqueous solution temperature is 23 ℃, treatment time is 10min, and then transferred into culture water to complete hatching.
Comparative example 15: 35min after fertilization, the hydrostatic pressure is 1200PSI, the action time is 3min, after hydrostatic pressure treatment, the fertilized eggs are subjected to chemical substance induction treatment, the fertilized eggs are immersed into 1.5mg/L of cytochalasin B aqueous solution for treatment, the cytochalasin B aqueous solution temperature is 23 ℃, the treatment time is 20min, and then the fertilized eggs are transferred into culture water to complete hatching.
Comparative example 16: 35min after fertilization, the hydrostatic pressure is 1200PSI, the action time is 3min, after hydrostatic pressure treatment, the fertilized eggs are subjected to chemical substance induction treatment, the fertilized eggs are immersed into 3.0mg/L of cytochalasin B aqueous solution for treatment, the cytochalasin B aqueous solution temperature is 23 ℃, the treatment time is 10min, and then the fertilized eggs are transferred into culture water to complete hatching.
The tetraploid proportion obtained in the above comparative examples was counted, and the tetraploid proportion produced in comparative example 14 was the highest and the tetraploid proportion was 17% in each comparative example, so that it was confirmed that the chemical induction treatment was a treatment of immersing fertilized eggs subjected to hydrostatic pressure treatment in 1.5mg/L of an aqueous solution of cytochalasin B at 23℃for 10 minutes, and then transferring the fertilized eggs into culture water to complete hatching.
(5) Determination of whether to perform temperature shock and chemical combination induction treatment:
comparative example 14: 35min after fertilization, the hydrostatic pressure is 1200PSI, the action time is 3min, after hydrostatic pressure treatment, the fertilized eggs are subjected to chemical substance induction treatment, the fertilized eggs are immersed into 1.5mg/L of cytochalasin B aqueous solution for treatment, the cytochalasin B aqueous solution temperature is 23 ℃, the treatment time is 10min, and then the fertilized eggs are transferred into culture water to complete hatching.
Comparative example 17: 35min after fertilization, the hydrostatic pressure is 1200PSI, the action time is 3min, after hydrostatic pressure treatment, the fertilized eggs are subjected to temperature shock and chemical substance combined induction treatment, the fertilized eggs are immersed into 1.5mg/L cytochalasin B aqueous solution for treatment, the cytochalasin B aqueous solution temperature is-2-2 ℃, the treatment time is 10min, and then the fertilized eggs are transferred into culture water to complete incubation.
Comparative example 18: 35min after fertilization, the hydrostatic pressure is 1200PSI, the action time is 3min, after hydrostatic pressure treatment, the fertilized eggs are subjected to temperature shock and chemical substance combined induction treatment, the fertilized eggs are immersed into 1.5mg/L cytochalasin B aqueous solution for treatment, the cytochalasin B aqueous solution temperature is-2-2 ℃, the treatment time is 20min, and then the fertilized eggs are transferred into culture water to complete incubation.
Comparative example 19: 35min after fertilization, the hydrostatic pressure is 1200PSI, the action time is 3min, after hydrostatic pressure treatment, the fertilized eggs are subjected to chemical substance induction treatment, the fertilized eggs are immersed in 1.5mg/L of cytochalasin B aqueous solution for treatment, the cytochalasin B aqueous solution temperature is 23 ℃, the treatment time is 20min, and then the fertilized eggs are transferred into culture water to complete hatching.
The tetraploid proportions obtained in the above comparative examples were counted, and comparative example 17 produced the highest tetraploid proportion of 18% in each comparative example. Therefore, the fertilized egg after hydrostatic pressure treatment is determined to be subjected to temperature shock and chemical substance combined induction treatment, wherein the temperature shock and chemical substance combined induction treatment is to immerse the fertilized egg after hydrostatic pressure treatment into 1.5mg/L cytochalasin B aqueous solution for 10min at the temperature of-2-2 ℃, and then transfer the fertilized egg into culture water to complete hatching.
(6) Determination of whether or not to perform the chemical induction treatment after the temperature shock and the chemical combination induction treatment, and the chemical induction treatment time:
comparative example 17: 35min after fertilization, the hydrostatic pressure is 1200PSI, the action time is 3min, after hydrostatic pressure treatment, the fertilized eggs are subjected to temperature shock and chemical substance combined induction treatment, the fertilized eggs are immersed into 1.5mg/L cytochalasin B aqueous solution for treatment, the cytochalasin B aqueous solution temperature is-2-2 ℃, the treatment time is 10min, and then the fertilized eggs are transferred into culture water to complete incubation.
Comparative example 20: 35min after fertilization, hydrostatic pressure of 1200PSI, action time of 3min, temperature shock and chemical substance combined induction treatment of fertilized eggs, treatment of the fertilized eggs by immersing the fertilized eggs in 1.5mg/L of cytochalasin B aqueous solution, treatment time of 10min at-2-2 ℃, chemical substance induction treatment, and incubation of the fertilized eggs in 1.5mg/L of cytochalasin B aqueous solution at 23-25 ℃ for 1h; and finally, transferring the fertilized eggs into culture water, and finishing incubation at the constant temperature of 23-25 ℃.
Comparative example 21: 35min after fertilization, the hydrostatic pressure is 1200PSI, the action time is 3min, after hydrostatic pressure treatment, the fertilized egg is subjected to temperature shock and chemical substance combined induction treatment, the fertilized egg is immersed into 1.5mg/L cytochalasin B aqueous solution for treatment, the temperature of the cytochalasin B aqueous solution is-2-2 ℃, and the treatment time is 10min; then carrying out chemical substance induction treatment, and transferring the fertilized eggs into 1.5mg/L cytochalasin B aqueous solution with the temperature of 23-25 ℃ for hatching for 2 hours; and finally, transferring the fertilized eggs into culture water, and finishing incubation at the constant temperature of 23-25 ℃.
The tetraploid proportions obtained in the above comparative examples were counted, and comparative example 20 produced the highest tetraploid proportion of 23% in each comparative example.
The tetraploid proportion of each comparative example is integrated, and the production method for producing the tetraploid culter ilishaeformis is finally determined:
(1) Artificial insemination of culter ilishaeformis:
the method comprises the steps of collecting mature eggs and semen after selective maturation of the culter ilishaeformis, placing the eggs in a drying container at 23-25 ℃, slowly adding the semen with the volume ratio of 1000 mu L to 100mL, performing dry fertilization, slowly operating to mix the semen and the eggs uniformly, then adding culture water at 23-25 ℃ which is 10 times of the egg volume, wherein the culture water meets the fishery water quality standard (GB 11607-1989), and slowly shaking to complete the insemination process of the semen and the eggs.
(2) Inducing tetraploid culter ilishaeformis:
when the fertilized ovum is 35min after insemination, placing the fertilized ovum into a hydrostatic press bin for hydrostatic pressure treatment, wherein the pressure is 1200PSI, and the treatment time is 3min; after hydrostatic pressure treatment, the fertilized eggs are subjected to temperature shock and chemical substance combined induction treatment, the fertilized eggs are immersed into 1.5mg/L cytochalasin B aqueous solution for treatment, the temperature of the cytochalasin B aqueous solution is-2-2 ℃, and the treatment time is 10min; then carrying out chemical substance induction treatment, and transferring the fertilized eggs into 1.5mg/L cytochalasin B aqueous solution with the temperature of 23-25 ℃ for hatching for 1h; and finally, transferring the fertilized eggs into culture water, and finishing incubation at the constant temperature of 23-25 ℃.
2. Identification of tetraploid Erythroculter ilishaeformis
(1) Identification of growth parameters
The body length and the body weight of the diploid Erythroculter ilishaeformis and the tetraploid Erythroculter ilishaeformis obtained by the production method of the tetraploid Erythroculter ilishaeformis under the same culture condition are measured, and experimental samples consist of 5 age groups of 2-6 ages, and the body weight and the body length parameters are shown in Table 1.
As can be seen from the data in Table 1, the diploid Erythroculter ilishaeformis and tetraploid Erythroculter ilishaeformis differ significantly in body length and body weight (P < 0.05). The length ratio of the 2-year-old diploid culter ilishaeformis to the tetraploid culter ilishaeformis is 1:1.53, weight ratio 1: the length ratio of the 1.42,4-age diploid culter ilishaeformis to the tetraploid culter ilishaeformis is 1:1.71, weight ratio 1:1.68. under the same culture conditions, the tetraploid culter ilishaeformis is obviously superior to diploid in terms of body length and body weight characteristics, and has faster growth speed and larger individual.
TABLE 1 diploid (2 n) Erythroculter ilishaeformis and tetraploid (4 n) Erythroculter ilishaeformis body length and body weight parameters
(2) Erythrocyte volume identification
Cell size is believed to be related to chromosome and DNA content, and in a single cell, because the number of tetraploid chromosomes is greater than that of diploid, the tetraploid cell volume is generally greater than that of diploid in order to maintain a constant nuclear mass fraction. The parameters of the diploid Erythroculter ilishaeformis and the red blood cells and the cell nuclei of the tetraploid Erythroculter ilishaeformis obtained by the production method of the tetraploid Erythroculter ilishaeformis under the same culture condition are measured, wherein the measurement method is to collect the blood of the diploid Erythroculter ilishaeformis and the tetraploid Erythroculter ilishaeformis, prepare blood smears, HE staining and observe under an optical microscope. The results of parameters of red blood cells and nuclei of diploid Erythroculter ilishaeformis and tetraploid Erythroculter ilishaeformis are shown in Table 2.
As can be seen from Table 2, the tetraploid Erythroculter ilishaeformis has values significantly higher than the diploid Erythroculter ilishaeformis. The length, the short diameter, the red cell area and the red cell volume of the tetraploid culter ilishaeformis are respectively 1.76 times, 1.65 times, 1.72 times and 1.72 times of that of the diploid culter ilishaeformis, the length, the short diameter, the nuclear area and the volume of the erythrocyte nuclei of the tetraploid culter ilishaeformis are respectively 1.81 times, 1.79 times, 1.84 times and 1.79 times of that of the diploid erythrocyte nuclei, and each parameter of the diploid erythrocyte culter ilishaeformis and the tetraploid erythrocyte culter ilishaeformis has obvious difference (P < 0.05). Wherein the diploid Erythroculter ilishaeformis erythrocyte volume range is 70.23+ -12.26 μm 3 The volume of the red blood cell nucleus is in the range of 56.67 plus or minus 6.14 mu m 3 Between them; the volume of the tetraploid culter erythrocyte is within 130.62 +/-9.57 mu m 3 The volume of the red blood cell nucleus is within the range of 101.44 +/-24.12 mu m 3 Between them; the volume ratio of the diploid Erythroculter ilishaeformis to the tetraploid Erythroculter ilishaeformis red cells is 1:1.86, and the volume ratio of the red cell nuclei is 1:1.79.
TABLE 2 erythrocyte and nuclear parameters of diploid (2 n) Erythroculter ilishaeformis and tetraploid (4 n) Erythroculter ilishaeformis
(3) DNA content identification
And (3) measuring the DNA content of the diploid Erythroculter ilishaeformis and the tetraploid Erythroculter ilishaeformis obtained by the tetraploid Erythroculter ilishaeformis production method under the same culture condition. The determination method is that tail vein blood is taken from the diploid culter ilishaeformis and tetraploid culter ilishaeformis, blood is taken from the tail vein: mixing 0.75% physiological saline=1:4 ratio, separating lymphocyte with lymphocyte separating liquid, dyeing PI in dark at 4deg.C for 30min, filtering with PARTEC ploidy tester with 400 mesh sieve, and performing ploidy identification. 100 samples (50 each of diploid Erythroculter ilishaeformis and tetraploid Erythroculter ilishaeformis) were tested and used as controls with chicken blood of known DNA content. The relative content of diploid Erythroculter ilishaeformis and tetraploid Erythroculter ilishaeformis DNA produced by the method is analyzed by flow cytometry to determine ploidy, and the flow analysis experimental results are shown in figure 1 and figure 2. The DNA content of the diploid Erythroculter ilishaeformis, tetraploid Erythroculter ilishaeformis and chickens is shown in Table 3, the DNA relative content of the diploid Erythroculter ilishaeformis is 55.43+/-11.52, the DNA relative content of the tetraploid Erythroculter ilishaeformis 112.03 +/-11.37, and the DNA content of the tetraploid Erythroculter ilishaeformis 2.02 times of that of the diploid Erythroculter ilishaeformis. Known chicken erythrocyte DNA content 2.3pg/N -1 The diploid somatic cell has an absolute DNA content of 2.25pg.N -1 The tetraploid Erythroculter ilishaeformis somatic cell absolute DNA content is 4.55pg. N -1 。
TABLE 3 DNA content of diploid (2 n) Erythroculter ilishaeformis, tetraploid (4 n) Erythroculter ilishaeformis and chickens
(4) Chromosome count identification
The diploid Erythroculter ilishaeformis and the tetraploid Erythroculter ilishaeformis obtained by the production method of the invention under the same culture condition are observed, the method is to prepare the metaphase of the chromosome by adopting a kidney cell in-vivo culture method, and the chromosome is stained by Giemsa and observed under a microscope. The results are shown in fig. 3 and 4, and the counting result shows that the number of the chromosomes of the diploid culter ilishaeformis is 2n=48, the number of the chromosomes of the tetraploid culter ilishaeformis is 4n=96, the number of the chromosomes of the tetraploid culter ilishaeformis 2.0 times that of the diploid culter ilishaeformis, and the number of the chromosomes accords with the ploidy relation.
(5) Gonadal tissue section identification
The diploid Erythroculter ilishaeformis and the gonadal tissue sections of the tetraploid Erythroculter ilishaeformis obtained by the production method of the tetraploid Erythroculter ilishaeformis under the same culture condition are observed, the gonadal tissue is fixed in 4% paraformaldehyde, dehydrated by a gradient ethanol solution, removed from xylene and embedded in paraffin, 5-6 mu m thick sections are prepared and stained with hematoxylin-eosin (H & E), and microscopic observation is carried out. The gonadal tissue section is shown in fig. 5 and 6, and the comparison of the sperm nest section of the 4-year-old tetraploid culter ilishaeformis with the sperm nest section of the diploid culter ilishaeformis shows that the sperm nest tissue structure is compact, a large number of differentiated germ cells are contained, the number of cells and mitochondria is normal, and no obvious difference exists between the two.
The identification method is summarized as follows: compared with diploid culter ilishaeformis, the tetraploid culter ilishaeformis obtained by the production method of the invention has obviously increased body length and weight, obviously increased area and volume of red blood cells, increased multiple of chromosomes, correspondingly increased content of DNA in cells, proportional cell volume to multiple of chromosomes, more compact testis tissue structure and data difference conforming to ploidy relation.
In the identification method of the tetraploid culter ilishaeformis, the growth parameter identification can be used as a preliminary screening method, the erythrocyte assay method can be used as a preferred method for identifying ploidy in a large scale, and the identification accuracy can be perfected by chromosome counting, DNA content and tissue section identification methods, so that the rapid and effective ploidy identification can be carried out on a large scale of fishes. The invention opens up a new technical approach for sex control of tetraploid culter ilishaeformis and polyploid breeding.
While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

Claims (5)

1. A method for producing tetraploid culter ilishaeformis, comprising the steps of:
(1) Artificial insemination of culter ilishaeformis: collecting mature eggs and semen of the culter ilishaeformis, placing the eggs in a drying container, slowly adding the semen, performing dry fertilization, slowly operating to uniformly mix the semen and the eggs, then adding culture water, and slowly shaking to complete the insemination process of the semen and obtain fertilized eggs;
(2) Inducing tetraploid culter ilishaeformis: placing the fertilized eggs obtained in the step (1) into a hydrostatic press bin, performing hydrostatic pressure treatment, performing temperature shock and cytochalasin B aqueous solution combined induction treatment on the fertilized eggs subjected to the hydrostatic pressure treatment, taking out the fertilized eggs, transferring the fertilized eggs into the cytochalasin B aqueous solution for chemical induction treatment, and finally transferring the fertilized eggs into culture water for hatching; the hydrostatic pressure treatment is carried out for 3min after insemination for 35min, wherein the pressure is 1200 PSI; the temperature shock and cytochalasin B aqueous solution combined induction treatment is that fertilized eggs are immersed into the cytochalasin B aqueous solution with the temperature of-2-2 ℃ and the concentration of 1.5mg/L for 10min; the chemical induction treatment is to incubate fertilized eggs in 1.5mg/L cytochalasin B aqueous solution at 23-25 ℃ for 1h.
2. The method of claim 1, wherein the volume ratio of semen to eggs in step (1) is 1000 μl:100 mL.
3. The process according to claim 1, wherein the temperature of the drying vessel in step (1) is 23 to 25 ℃.
4. The method according to claim 1, wherein the temperature of the water for cultivation in the step (1) is 23-25 ℃, and the addition amount is 10 times the egg volume.
5. The method of claim 1, wherein the incubating in the aquaculture water of step (2) is performed with aquaculture water Shui Wenheng at 23-25 ℃.
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